Annals of Agricultural and Environmental Medicine 2016, Vol 23, No 4, 666–670 www.aaem.pl ORIGINAL ARTICLE

Diagnostics of intestinal parasites in light microscopy among the population of children in eastern Afghanistan Krzysztof Korzeniewski1, Agata Smoleń2, Alina Augustynowicz1, Anna Lass3 1 Department of Epidemiology and Tropical Medicine, Military Institute of Medicine, Gdynia, Poland 2 Department of Epidemiology and Methodology of Clinical Research, Medical University, Lublin, Poland 3 Department of Tropical Parasitology, Institute of Maritime and Tropical Medicine, Medical University, Gdańsk, Poland Korzeniewski K, Smoleń A, Augustynowicz A, Lass A. Diagnostics of intestinal parasites in light microscopy among the population of children in eastern Afghanistan. Ann Agric Environ Med. 2016; 23(4): 666–670. doi: 10.5604/12321966.1226864 Abstract Objectives. The Afghans, living in poor socioeconomic conditions, are estimated to be a community with a high rate of intestinal parasitic infections. The aim of the study was to estimate the prevalence and species of intestinal parasites among children’s population in eastern Afghanistan and to present the methods of optimizing the techniques for identification of pathogens in light microscopy. The research was carried out as a part of humanitarian project Capacity building of health care system in Ghazni Province. Materials and method. The study involved 500 children aged 7–18 attending the Share Kona and the Khuija Ali High Schools in Ghazni, eastern Afghanistan in the period November 2013-April 2014. Three stool samples were collected from each patient at 2-day intervals, the samples were fixed in 10% formalin, transported to the Military Institute of Medicine in Poland, where they were pooled and examined using five different diagnostic methods in light microscopy (direct smear in Lugol’s solution, Fülleborne’s flotation, decantation in distilled water, Kato-Miura thick smear, and DiaSys/PARASYS sedimentation system). Results. Pathogenic intestinal parasites were detected in 217 patients (43.4%), with the most common (35.3%), Giardia intestinalis (31.1%), and nana (15.7%). The use of direct smear method allowed for the detection of intestinal parasites in 161 individuals. The application of four following testing methods has improved the detection rates of infected patients by 11.2%. Conclusions. The variety of detected intestinal pathogens in examined children’s population has required the use of combination of multiple diagnostic methods in light microscopy, and finally improved the detection rates of intestinal parasites and helped eliminate infections with , cestodes, trematodes, and protozoa using appropriate treatment in the study population. Key words Afghanistan, intestinal parasites, light microscopy

INTRODUCTION Afghanistan was performed in 2002 by the German Armed Forces health service stationed in Kabul. Among 217 local The Afghans, living in poor socio-economic conditions, are workers from the international military base (kitchen aids, estimated to be a community with a high rate of intestinal translators, housekeeping personnel), 64% were infected parasitic infections. Unfortunately, reports concerning with intestinal helminths and protozoa, with Ascaris population morbidity rates often are not confirmed by lumbricoides predominance observed (22.1%) [4]. In 2003, laboratory tests. Afghanistan is one of 57 countries associated World Health Organization workers performed a screening with the Organization of the Islamic Cooperation, with examination of stool samples taken from 1,001 children aged no data available on the prevalence of intestinal parasitic 8–15 in 4 provinces of the country. The results confirmed the infections among its inhabitants [1]. In literature, there is occurrence of intestinal helminthiases in 47% of the children little information concerning infections caused by intestinal with the predominance of Ascaris lumbricoides [5]. parasites in Afghan refugees in the USA and Europe. In Since 2002, the military health service of the Polish Armed the 1980s, an examination of 51 Afghans performed in Forces has been stationed in eastern Afghanistan. Under USA showed that 32% of them were infected with Ascaris the framework of humanitarian aid, the Polish doctors and lumbricoides, Giardia intestinalis, Entamoeba histolytica, diagnosticians were mandatorily assigned to a Forward and [2]. According Operating Base in Ghazni, where they carried out the to studies performed on 5,928 of Afghan refugees between collection of biological samples for parasitological research 1997 – 2003 in Sweden, 225 were infected with Giardia with the aim of detecting and eliminating intestinal parasitic intestinalis [3]. One of a few parasitological researches in infections in the local population. There were some diagnostic dilemmas with reference to stool examination at the stage of morbidity control, because Address for correspondence: Krzysztof Korzeniewski, Department of Epidemiology and Tropical Medicine, Grudzinskiego 4, 81-103 Gdynia 3, Poland infections with multiple species are the norm rather than E-mail: [email protected] exception. There is a need for well-trained laboratory Received: 27 September 2015; accepted: 26 September 2016 diagnosticians and quality-control measures to ascertain Annals of Agricultural and Environmental Medicine 2016, Vol 23, No 4 667 Krzysztof Korzeniewski, Agata Smoleń, Alina Augustynowicz, Anna Lass. Diagnostics of intestinal parasites in light microscopy among the population of children… accurate, species-specific parasites diagnosis [6]. Another smeared on the slide. Next, a cover slide is placed on top of diagnostic problem could be the long time required for the preparation. This method allows for an initial analysis collecting stool specimens in the field, transferring them of non-concentrated material under correct magnification to the laboratory and preparing the slides for examination, of objective (×10, then ×40). which can result in notable underestimates of intestinal parasites burdens [7]. For diagnosing intestinal parasites Fülleborne’s flotation. Approximately 2 g of stool is mixed with light microscopy, fast, direct methods such as faecal with saturated NaCl solution in a test tube. Next, NaCl smear and Kato-Katz thick smear are most widely used solution is added to the top of the tube. A covered slide is worldwide [8]. For the detection of the soil-transmitted placed on top of the tube and in contact with the suspension. helminths (Ascaris lumbricoides, Trichuris trichiura, After 20 minutes, the cover slide is removed with tweezers /) the World and placed wet side down on a slide. The preparation is ready Health Organization recommends using the Kato-Katz for microscopic examination (objective ×10 magnification). method based on duplicate slides [9]. Other commonly used This method is best for examination of helminth eggs which methods include direct smear, formol-ether concentration, are lighter than the NaCl solution and are pushed upwards FLOATAC and Mini-FLOATAC. All of these techniques and connect to the cover slide. rely on visual examination of a small sample of stool to determine the presence and number of helminth eggs [10]. Decantation in distilled water. Approximately 2 g of stool is Other modern techniques employed for the detection of life mixed thoroughly with a small amount of distilled water in cycle stages of intestinal parasites (helminths and protozoa), a test tube. Next, water is added to the top of the tube and which were used in the presented study, include PARASYS mixed again. The supernatant is decanted after 30 minutes and Fülleborne’s flotation. and another portion of water is added. The procedure was repeated until a clear supernatant is obtained, generally 3–4 times. The sediment is then placed on a slide and stained OBJECTIVES with Lugol’s solution, ready for microscopic examination (objective ×40 magnification). This method is very useful The aim of the study was to estimate the prevalence and for examination of protozoan parasites. species of intestinal parasites among the population of children in eastern Afghanistan, and to present the methods Kato-Miura thick smear. This method is used to prepare a of optimizing the techniques for identification of pathogens thick smear using a colouring Kato reagent for observation in light microscopy. The research was carried out as a part in light microscopy. It is effective in screening for the eggs of of a humanitarian project Capacity building of health care intestinal parasites, especially Ascaris lumbricoides. The Kato system in Ghazni Province, financed by the Polish Ministry reagent contains of 100 ml distilled water, 100 ml of glycerin of National Defence and realized by diagnosticians of the and 3 drops of 3% malachite green. Strips of cellophane must Epidemiology and Tropical Medicine Department in Military be soaked in a well mixed Kato reagent for at least 24 hours. Institute of Medicine in Gdynia, Poland. To examine the specimen, a bead of stool is placed onto the slide and covered with a cellophane strip. Next, it is turned over and the top of the preparation is pressed onto a paper MATERIALS AND METHODS filter and left for one hour at room temperature. The sample is then ready for examination (objective ×10 magnification). Study population. In total, 500 Afghan children aged 7–18, attending the Share Kona and Khuija Ali High Schools (most DiaSys/PARASYS sedimentation system. PARASYS is a lab of students in both schools) in Ghazni, the capital city of station equipped with a dual-stream tube system which Ghazni Province, were surveyed in the period November allows a thorough examination of the concentrated material 2013-April 2014. Examined students were representatives of (making it possible to observe the morphology of parasites the children inhabiting eastern Afghanistan. in the suspended state, and minimize the presence of faeces and other solids). The closed (isolated) process eliminates any Sample collection. Three stool samples were collected contact with the laboratory sample. There are 4 steps in the from each child at 2-day intervals, the samples were fixed sample preparation process. First, the test tube is filled with in 10% formalin, and transported to the Military Institute of 2.5 ml of 10% formalin. Then, a drop of surfactant, usually Medicine in Poland where they were pooled and examined Triton X, is added to the tube. Next, 1ml of ethyl acetate using 5 different diagnostic methods in light microscopy: is added to the mix. The last step is adding approximately direct smear in Lugol’s solution, Fülleborne’s flotation, 0.5 g of stool to the test tube and vortexing it thoroughly in decantation in distilled water, Kato-Miura thick smear and order to obtain a homogeneous solution. This solution needs the DiaSys/PARASYS sedimentation system. Thus, 2,500 to be centrifuged at 1,000 RCF for 1 minute. The obtained parasitological tests were performed in total. sediment is ready for microscopic examination (objective ×20 and ×40 magnification). Laboratory procedures. The diagnostics of intestinal parasites was performed by light microscopy using 5 stool Statistical analysis. Analysis was performed using the testing methods: statistical programme StatSoft Inc. (2011) STATISTICA (data analysis software system) version 10.0. www.statsoft.com (SN Direct smear in Lugol’s solution. Approximately 2 mg of JGNP3087539302AR-E) and Excel. The qualitative variables stool is collected with a glass rod and applied onto a slide, were presented with the use of count and percentage. Chi- a drop of Lugol’s solution is then added and the material squared tests for independence were used for qualitative 668 Annals of Agricultural and Environmental Medicine 2016, Vol 23, No 4 Krzysztof Korzeniewski, Agata Smoleń, Alina Augustynowicz, Anna Lass. Diagnostics of intestinal parasites in light microscopy among the population of children… variables (with the use of Yates correction for cell counts infections), Giardia intestinalis (31.1%), and Hymenolepis below 10, with check of Cochrane’s conditions or with Fisher’s nana (15.7%) (Tab. 1). 58 of the tested children were diagnosed exact test respectively). A sign test was used to evaluate with multiple infections, mainly +giardiasis, differences between particular methods (direct smear vs. all ascariasis+, and giardiasis+hymenolepiasis. 5 methods). In all the calculations, the statistical significance The use of the basic direct smear method resulted in level of p=0.05 was used. detection of intestinal parasites in 161 individuals. The application of another 4 testing methods improved the detection rates of infected patients by 11.2 % (217 infected RESULTS children). The number of infections increased from 202 when the direct smear method was used, to 286 when all 5 diagnostic Among 500 of the study subjects – Afghan students of the methods were applied (Tab. 2). The percentage of infections Share Kona and the Khuija Ali High Schools in Ghazni, detected with the use of all 5 methods in comparison to the eastern Afghanistan – pathogenic intestinal parasitic application of a single method (direct smear) was significantly infections were detected in 217 children (prevalence of higher in cases of Ascaris lumbricoides (p=0.000001), infection 43.4%). The most common pathogens detected in Hymenolepis nana (p=0.00006), and Giardia intestinalis the studied individuals were Ascaris lumbricoides (35.3% of infection (p=0.0002) (Tab. 3).

Table 1. Intestinal parasitic infections in selected children in Ghazni, eastern Afghanistan, November 2013-April 2014 (n=500) Number of infec­tions Percent­age Percent­age of infected Percent­age of tested Intestinal parasites Treat­ment (n=286) of infec­tions children (n=217) children (n=500) Nematodes 121 42.3 55.8 24.2 Ascaris lumbricoides 101 35.3 46.5 20.2 Enterobius vermicularis 12 4.2 5.5 2.4 albendazole Ancylostoma duodenale/ Necator americanus 4 1.4 1.8 0.8 albendazole Strongyloides stercoralis 1 0.35 0.5 0.2 albendazole Trichuris trichiura 1 0.35 0.5 0.2 albendazole spp. 2 0.7 0.9 0.4 albendazole Cestodes 60 20.9 27.6 12.0 Hymenolepis nana 45 15.7 20.7 9.0 2 0.7 0.9 0.4 praziquantel Taenia spp. 13 4.5 6.0 2.6 praziquantel Trematodes 8 2.8 3.6 1.6 6 2.1 2.8 1.2 praziquantel 2 0.7 0.9 0.4 triclabendazole Protozoa 97 33.9 44.7 19.4 Giardia intestinalis 89 31.1 41.0 17.8 metronidazole Entamoeba histolytica s. l. 8 2.8 3.6 1.6 metronidazole Number of infected children 217 100.0 100.0 43.4

Table 2. Identification of intestinal parasites using 5 diagnostic methods in light microscopy in selected children in Ghazni, eastern Afghanistan, November 2013-April 2014 (n=500)

Direct smear Decantation Fülleborne’s Kato-Miura TOTAL DiaSys/ PARASYS in Lugol’s solution in distilled water flotation thick smear (5 methods} Intestinal parasites Number Number Number Number Number Number of infections of infections of infections of infections of infections of infections n=202 (%) n=171 (%) n=84 (%) n=105 (%) n=155 (%) n=286 (%) Nematodes Ascaris lumbricoides 75 (15.0) 64 (12.8) 48 (9.6) 80 (16.0) 55 (11.0) 101 (20.2) Enterobius vermicularis 6 (1.2) 2 (0.4) 5 (1.0) 0 (0.0) 3 (0.6) 12 (2.4) Ancylostoma duodenale/ Necator 2 (0.4) 0 (0.0) 0 (0.0) 1 (0.2) 3 (0.6) 4 (0.8) americanus Strongyloides stercoralis 1 (0.2) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.2) Trichuris trichiura 0 (0.0) 0 (0.0) 1 (0.2) 1 (0.2) 0 (0.0) 1 (0.2) Trichostrongylus spp. 0 (0.0) 0 (0.0) 2 (0.4) 0 (0.0) 0 (0.0) 2 (0.4) Cestodes Hymenolepis nana 27 (5.4) 21 (4.2) 20 (4.0) 19 (3.8) 22 (4.4) 45 (9.0) Hymenolepis diminuta 2 (0.4) 1 (0.2) 1 (0.2) 0 (0.0) 1 (0.2) 2 (0.4) Taenia spp. 7 (1.4) 5 (1.0) 2 (0.4) 1 (0.2) 1 (0.2) 13 (2.6) Trematodes Dicrocoelium dendriticum 3 (0.6) 1 (0.2) 1 (0.2) 2 (0.4) 1 (0.2) 6 (1.2) Fasciola hepatica 1 (0.2) 1 (0.2) 0 (0.0) 0 (0.0) 0 (0.0) 2 (0.4) Protozoa Giardia intestinalis 75 (15.0) 72 (14.4) 4 (0.8) 0 (0.0) 67 (13.4) 89 (17.8) Entamoeba histolytica s. l. 4 (0.8) 3 (0.6) 0 (0.0) 1 (0.2) 3 (0.6) 8 (1.6) Number of infected children 161 143 79 99 142 217 Annals of Agricultural and Environmental Medicine 2016, Vol 23, No 4 669 Krzysztof Korzeniewski, Agata Smoleń, Alina Augustynowicz, Anna Lass. Diagnostics of intestinal parasites in light microscopy among the population of children…

Table 3. Identification of intestinal parasites using direct smear and all and malnutrition [16]. The diagnostics of intestinal parasites 5 diagnostic methods in light microscopy in selected children in Ghazni, is based mainly on light microscopy as a gold standard, eastern Afghanistan, November 2013-April 2014 (n=500) which allows for the detection of different stages of intestinal Direct smear in TOTAL parasites life cycles (cyst, oocyst, trophozoite, larva, egg) [17]. Lugol’s solution (5 methods} Although stool examination provides an acceptable measure Intestinal parasites Number Number p-value of the stage of infection in highly endemic areas, there was of infections of infections originally hope that antigen detection would do the same n=202 (%) n=286 (%) at the other end of the control spectrum (i.e. in areas of Nematodes very low endemicity). However, this has not been validated Ascaris lumbricoides 75 (15.0) 101 (20.2) 0.000001 because antigen-detection techniques are only marginally Enterobius vermicularis 6 (1.2) 12 (2.4) 0.02 more sensitive than stool examination. Although serology Ancylostoma duodenale/ and microscopy are complementary, it cannot be emphasized 2 (0.4) 4 (0.8) 0.48 Necator americanus enough that the integration of serological methods into Strongyloides stercoralis 1 (0.2) 1 (0.2) 1 national control programmes requires the development of accurate, methodologically standardized and easily Trichuris trichiura 0 (0.0) 1 (0.2) 1 applicable assays for the detection of both specific antibodies Trichostrongylus spp. 0 (0.0) 2 (0.4) 0.48 and antigens [6]. Successful detection of parasites with light Cestodes microscopy is strongly associated with the diagnostic methods Hymenolepis Nana 27 (5.4) 45 (9.0) 0.00006 used, and a well-trained microscopist. The success rate will be Hymenolepis diminuta 2 (0.4) 2 (0.4) 1 rarely higher than 30% if the direct smear method is used by a person who is inexperienced in parasitological diagnostics. Taenia spp. 7 (1.4) 13 (2.6) 0.04 In this study, a direct smear method was performed by well- Trematodes trained parasitologists in the Department of Epidemiology Dicrocoelium dendriticum 3 (0.6) 6 (1.2) 0.25 and Tropical Medicine, where the index of detection for this Fasciola hepatica 1 (0.2) 2 (0.4) 1 basic method amounted to 74.2%. An important diagnostic Protozoa problem in light microscopy could be the long time-lapse between collecting the stool specimens in the field, to Giardia intestinalis 75 (15.0) 89 (17.8) 0.0002 examination of biological samples in the laboratory. This can Entamoeba histolytica s. l. 4 (0.8) 8 (1.6) 0.13 result in the underestimation of parasitic infections as a result Number of infected children 161 217 of the influence of the preservatives used for stool fixation. The limitation of this study was the 2–4 week period between collection of samples in Afghanistan and examination of the The most effective examination among the added 4 fixed biological material after its transportation to Poland. testing methods was the Kato-Miura thick smear in Parasitological laboratories often use concentration methods, detection of Ascaris lumbricoides and Dicrocoelium which significantly increase the detection limit of intestinal dendriticum, Fülleborne’s flotation in the detection of parasites. Using several diagnostic methods is reasonable Enterobius vermicularis, DiaSys/PARASYS in the detection for screening studies of both populations characterized by of Hymenolepis nana and Ancylostoma duodenale/Necator a low infection rate (detection of single pathogens), and americanus, and decantation in distilled water in the detection communities where infections occur more often (detection of Giardia intestinalis and Taenia spp. The variety of detected of all pathogens in multiple infections) [18]. The inhabitants intestinal pathogens in the population of examined children of Afghanistan are an example of a community with a high required the use of a combination of multiple diagnostic prevalence of intestinal multiple infections. It seems that methods in light microscopy. This combined application single diagnostic methods and single-dose chemotherapy improved the detection rates of intestinal pathogens and (antiparasitic treatment with albendazole 400 mg or helped eliminate infections using appropriate treatment mebendazole 500 mg, common in the Third World) may (albendazole, metronidazole, praziquantel) in the study prove ineffective in the elimination of intestinal parasites population (Tab. 1). in the local population [19]. The application of multiple testing methods can improve not only the detection rates of infected patients (in this study by 11.2%) but can also DISCUSSION help to eliminate multiple co-infections with helminths and protozoa using the right treatment. The choice of a diagnostic Intestinal parasites pose a significant challenge for public assay should be governed by the objective of the activity. A health and especially for children living in developing global assessment of diagnostic test sensitivities and their countries [11, 12]. Infections with these parasites are highly extent of variation is required to investigate the suitability endemic in populations with a low socio-economic status of diagnostic tools for different control programmes [20]. and living in poor hygiene environments [13, 14]. Screening the Afghan community to optimize the process In this study, intestinal parasites were detected in 43.4% of of identification and elimination of intestinal parasites was Afghan children, and in other reports concerning inhabitants one of the humanitarian control programmes carried out in of Afghanistan in 47–64% [4,5]. Parasitic infections can cause developing countries. serious health problems, such as iron deficiency anaemia and growth retardation [15]. The spectrum of clinical manifestations of these infections is wide and includes asymptomatic carriage, long-lasting , stomach ache 670 Annals of Agricultural and Environmental Medicine 2016, Vol 23, No 4 Krzysztof Korzeniewski, Agata Smoleń, Alina Augustynowicz, Anna Lass. Diagnostics of intestinal parasites in light microscopy among the population of children…

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